AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity

Ngoc Tam Tran; Cheryl Heiner; Kristina Weber; Michael Weiand; Daniella Wilmot; Jun Xie; Dan Wang; Alexander Brown; Sangeetha Manokaran; Qin Su; Maria L. Zapp; Guangping Gao; Phillip W.L. Tai

Molecular Therapy: Methods & Clinical Development (Sep 2020)

AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity

Ngoc Tam Tran,
Cheryl Heiner,
Kristina Weber,
Michael Weiand,
Daniella Wilmot,
Jun Xie,
Dan Wang,
Alexander Brown,
Sangeetha Manokaran,
Qin Su,
Maria L. Zapp,
Guangping Gao,
Phillip W.L. Tai

Affiliations

Ngoc Tam Tran: Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, 01605, USA
Cheryl Heiner: Pacific Biosciences, Inc., Menlo Park, CA 94025, USA
Kristina Weber: Pacific Biosciences, Inc., Menlo Park, CA 94025, USA
Michael Weiand: Pacific Biosciences, Inc., Menlo Park, CA 94025, USA
Daniella Wilmot: Program in Molecular Medicine and Center for AIDS Research, University of Massachusetts Medical School, Worcester, MA 01605, USA
Jun Xie: Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, 01605, USA; Viral Vector Core, University of Massachusetts Medical School, Worcester, MA 01605, USA
Dan Wang: Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, 01605, USA
Alexander Brown: Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, 01605, USA
Sangeetha Manokaran: Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Viral Vector Core, University of Massachusetts Medical School, Worcester, MA 01605, USA
Qin Su: Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Viral Vector Core, University of Massachusetts Medical School, Worcester, MA 01605, USA
Maria L. Zapp: Program in Molecular Medicine and Center for AIDS Research, University of Massachusetts Medical School, Worcester, MA 01605, USA
Guangping Gao: Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, 01605, USA; Viral Vector Core, University of Massachusetts Medical School, Worcester, MA 01605, USA; Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, 01605, USA; Corresponding author: Guangping Gao, Horae Gene Therapy Center, University of Massachusetts Medical School, 386 Plantation Street, Worcester, MA 01605, USA.
Phillip W.L. Tai: Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, 01605, USA; Corresponding author: Phillip W.L. Tai, Horae Gene Therapy Center, University of Massachusetts Medical School, 386 Plantation Street, Worcester, MA 01605, USA.

Journal volume & issue: Vol. 18
pp. 639 – 651

Abstract

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The gene therapy field has been galvanized by two technologies that have revolutionized treating genetic diseases: vectors based on adeno-associated viruses (AAVs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene-editing tools. When combined into one platform, these safe and broadly tropic biotherapies can be engineered to target any region in the human genome to correct genetic flaws. Unfortunately, few investigations into the design compatibility of CRISPR components in AAV vectors exist. Using AAV-genome population sequencing (AAV-GPseq), we previously found that self-complementary AAV vector designs with strong DNA secondary structures can cause a high degree of truncation events, impacting production and vector efficacy. We hypothesized that the single-guide RNA (sgRNA) scaffold, which contains several loop regions, may also compromise vector integrity. We have therefore advanced the AAV-GPseq method to also interrogate single-strand AAV vectors to investigate whether vector genomes carrying Cas9-sgRNA cassettes can cause truncation events. We found that on their own, sgRNA sequences do not produce a high degree of truncation events. However, we demonstrate that vector genome designs that carry dual sgRNA expression cassettes in tail-to-tail configurations lead to truncations. In addition, we revealed that heterogeneity in inverted terminal repeat sequences in the form of regional deletions inherent to certain AAV vector plasmids can be interrogated.

Published in Molecular Therapy: Methods & Clinical Development

ISSN: 2329-0501 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Biology (General): Genetics; Science: Biology (General): Cytology
Website: http://www.cell.com/molecular-therapy-family/methods/latest-content

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